To determine the mechanism of visual information processing in Drosophila, we initiated a project aimed at mapping the connection patterns and function of visual circuits. Because of the vast complexity in the neuron subtypes and their interconnections in this system, we applied a divide and conquer strategy. First, we identify key neuron subtypes and their potential synaptic partners based on their unique axonal and dendritic patterns. Second, we subdivide these neurons based on their molecular properties, especially their use of neurotransmitters and receptors. Finally we determine their functions by inactivating or restoring their synaptic function and examining the behavioral consequences. We have chosen to focus on the first-order interneuorns in the medulla because (i) they process well-defined visual information directly from R7/R8 photoreceptors, (ii) they are likely involved in color-vision, and (iii) they are most relevant to our developmental studies described above.[unreadable] We reason that because Drosophila photoreceptor neurons are histaminergic neurons, the first-order interneurons must express the histamine receptor Ort in order to respond to the photoreceptors. We identified the ort promoter region and generated an ort-Gal4 driver. The ort promoter-Gal4 driver labels subsets of lamina and medulla neurons, which are likely direct synaptic targets of R1-6 and R7/8, respectively. In an electron-microscopic (EM) study, we confirmed that these ort (+) medulla neurons indeed form synaptic connections with R7 and R8 axonal termini. Furthermore, expressing the ort gene under the control of ort-Gal4 completely rescues the electrophysiology and behavioral defects of the ort mutants, indicating that the ort-Gal4 construct faithfully recapitulates the endogenous ort expression pattern.[unreadable] We performed single-cell analyses to identify the neuron subtypes that express Ort. Based on approximately 300 single-cell clones, we found that the ort-Gal4 labeled three types of lamina neurons (L1-3) as well as eight subtypes of medulla neurons. In particular, our observation that ort-Gal4 driver labeled L1-3 is in accord with a previous EM reconstruction study and validates our approach. We have chosen to focus on Tm5, Tm9 and Tm20 because (i) they were labeled with high frequency and therefore are likely present in every medulla column, and (ii) they extend dendritic processes in M6 (Tm5) and M3 (Tm5/9/20) and therefore might receive input from R7 and R8 (or L3) directly. [unreadable] The ort-Gal4 driver allows us to manipulate the first-order interneurons as a group but not individual subtypes. To do so, we employed the split-Gal4 system to restrict the Gal4 activity to different subclasses, which use distinct neurotransmitters. Specifically, we combine the ort promoter with promoters of specific neurotransmitters. We found that the combination of ort-AD-Zip and Chat-Gal4-DBD-Zip (which is expressed in Ort(+) cholinergic neurons) labeled two known subsets of Ort(+) neurons, including Tm9 and Tm20. In addition, we generated an AD-Zip enhancer trap vector and performed P-element swap to substitute Ok371vGlut enhancer trap, which labels glutaminergic neurons. The combinatorial driver composed of OK371-AD-Zip and ort-DBD-Zip labels a different subset of Ort(+) neurons, including a subclass of Tm5. These results indicate that the split-Gal4 system is suitable for manipulating distinct subsets of Ort(+) neurons. [unreadable] To determine the function of each neuron subtypes in color vision, we manipulate the activity of single or combinations of Ort(+) neuron subtypes and test whether specific Ort(+) subtypes are required or sufficient for color-discriminating behavior. We have previously found that flies have an innate preference for shorter wavelength (UV>>Blue>Green). In forced two-choice tests under a high green/UV light intensity ratio (1:150), over 85% of wild-type flies still preferred UV over green light, while ort mutant flies chose two light sources with a similar frequency. Conversely, mutant flies bearing ort->Shits1 lost their preference to UV at a non-permissive, but not permissive, temperature. These results indicate that Ort(+) neurons are functionally required for flies innate preference of UV. Our preliminary revealed that the loss of innate preference for UV in ort mutants can be rescued using the combinatorial driver of OK371 and ort driving the expression of the Ort gene. This result suggests that the Tm5 subtype mediates innate UV preference, presumably by relaying signals from the UV-sensitive R7s to the higher optic ganglion lobula.